PROJECT SUMMARY Hundreds of human proteins contain prion-like domains (PrLDs), defined as domains with compositional similarity to the yeast prion domains. In recent years, a growing number of PrLDs in various organisms have been shown to form functional assemblies that regulate cellular activities. Additionally, mutations in PrLDs have been linked to various degenerative disorders, including amyotrophic lateral sclerosis and frontotemporal dementia. Disease-associated mutations tend to increase the aggregation propensity of the PrLDs. This observation has led to the hypothesis that many PrLDs are designed to mediate dynamic reversible interactions involved in cellular regulation, and that mutations stabilize these interactions, resulting in dysregulation of the underlying cellular processes. Despite the importance of PrLDs in both normal physiology and pathology, numerous basic questions remain about the regulation of functional PrLD aggregation, and the relationship between functional and pathogenic aggregation. My lab has been a leader in efforts to define the sequence basis for aggregation of PrLDs. During the next 5 years, we will build on this work, focusing on three major areas: 1) We will define the sequence determinants of PrLDs that mediate the formation of dynamic functional assemblies. In recent years, my lab and others have made substantial progress in defining how the amino acid sequence of PrLDs affects formation of stable amyloid aggregates, but much less is known about the sequence determinants of the dynamic multi-component assemblies formed by some PrLDs. 2) We will examine the relationship between the dynamic functional assemblies formed by many PrLDs and the more stable aggregates seen in various diseases. 3) We will identify additional PrLDs involved in functional protein assemblies, and explore their mechanism of action. While PrLDs are highly over-represented in eukaryotic genomes, the functions of only a small subset of these PrLDs is known. We have identified two new PrLD- containing proteins involved in regulation of cellular stress responses. Therefore, we will characterize the mechanism of action of these two proteins, and screen for other functional PrLDs. Collectively, these studies will provide insight into the role of PrLDs in biological regulation and disease.